Project summary ?Evolutionary innovation? refers to the origin of entirely new traits, as opposed to the modification of existing traits. Although such novelties are relatively rare, all the complexity and diversity of life has ultimately been shaped by evolutionary innovations that occurred in a nested pattern, with every innovation dependent on many earlier novelties. Despite their critical importance for everything from geological nutrient cycles that support all life on Earth to human sentience that distinguishes us from our closest relatives, the molecular mechanisms of evolutionary innovations are understood far less than the evolution of existing traits. This is true at all levels of biological organization, from single molecules to the most complex features of animal form and function. We understand the evolution of existing organs and cell types better than the origin of new ones; quantitative variation in gene expression has been explored in much greater detail than the origin of novel regulatory pathways; much more is known about the evolution of existing genes than about the origin of new genes, and so on. It is this gap in our knowledge that motivates our work. To achieve a comprehensive understanding of evolutionary innovations, it is necessary to connect novelties at all levels of biological organization: from new functional elements in the genome, to new genetic pathways, to new morphological structures. Research in our lab will advance in three directions, using the Drosophila model system. First, we will identify the molecular mechanisms responsible for the origin of new morphological structures that evolved recently within Drosophila. We will identify the DNA sequences that gave rise to phenotypic innovations, and reconstruct the cell differentiation pathways than translate these changes into novel morphologies. Second, we will examine the genomic mechanisms that qualitatively remodel the gene expression profiles of different organs and cell types, and quantify the relative contributions of each type of genomic change to the turnover of genes expressed in each tissue. We will test whether the regulatory circuits that control gene expression evolve predominantly by incorporating individual genes, or by recruitment of larger genetic modules. Third, we will identify the molecular changes responsible for the origin of new regulatory elements that control gene expression from non-functional ancestral sequences. By focusing on novel regulatory elements that evolved within natural populations of a single species, we will reconstruct the series of mutations that create new functional elements in the genome, and elucidate the impact of these mutations on gene regulation. Together, these approaches will promote a deep mechanistic understanding of evolutionary innovations.